Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies

T cell–bispecific antibodies (BsAbs) couple cytotoxic T lymphocytes to tumor cells, inducing their destruction. Although there are more than 60 classes of BsAbs in development, the relative importance of parameters such as interdomain spacing or spatial configuration is largely unknown. Here, we dissected a symmetric dual bivalent BsAb platform (IgG-[L]-scFv: antitumor IgG with anti-CD3 scFv fused to the light chains) to explore the importance of valency and spatial configuration for BsAb-induced T cell cytotoxicity. Our results revealed that placing tumor and T cell binding domains on the same side of a BsAb (cis-configuration) elicited substantially stronger antitumor activity, in vitro and in vivo, compared to positioning them on opposite sides (trans-configuration). Moreover, using two cis-modules in the same BsAb further improved cytotoxicity (up to 2000-fold). In addition, separating antigen-binding components with a single Ig domain (CL) markedly enhanced cytokine release and in vivo tumor responses compared to smaller (G4S1) or larger (CH1-CH2-CH3) spacers. These findings provide guidelines for improving BsAb function and highlight the importance of spatial configuration and dual bivalency as development parameters.

Download Full-text

Evaluation of monovalent versus biparatopic CD3xPSMA bispecific antibodies for t-cell mediated killing of prostate tumor cells with minimal cytokine release.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e16519 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e16519-e16519

Author(s):

Ben Buelow ◽

Starlynn Clarke ◽

Kevin Dang ◽

Jacky Li ◽

Chiara Rancan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

T Cell Activation ◽

Cell Activation ◽

Bispecific Antibodies ◽

Cytokine Release ◽

Binding Domains ◽

Prostate Tumor Cells

e16519 Background: Castration resistant prostate cancer (CRPC) remains an incurable disease and new treatments are needed. Therapies directed against Prostate specific membrane antigen (PSMA) -such as radiolabeled antibodies, chimeric antigen receptor T cells (CAR-Ts) and T-cell engaging bispecific antibodies (T-BsAbs)- have shown promising efficacy but also induce significant toxicity. In particular T-cell redirection leads to efficient killing of tumor cells but induces cytokine release-related toxicities. We have developed a panel of monovalent and biparatopic CD3xPSMA bispecific antibodies that eliminate prostate tumor cells while minimizing cytokine release. Methods: Antibodies targeting CD3 and PSMA were generated in transgenic rats (UniRat™, OmniFlic™) followed by deep sequencing of the antibody repertoire from draining lymph nodes in immunized animals, and high-throughput gene assembly/expression. PSMA x CD3 T-BsAbs were assembled and evaluated for stability, pharmacokinetics, and T cell activation and ability to eliminate PSMA+ tumor cells in vitro and in vivo. Results: Bispecific CD3xPSMA Abs. incorporating either monovalent or biparatopic anti-PSMA binding domains activated T-cells in the presence of PSMA (plate-bound or cell surface), while no T cell activation occurred in the absence of either PSMA antigen or bispecific antibody. Potent/selective cytotoxicity against PSMA+ cells was observed in co-cultures of primary human T cells and tumor cells treated with CD3xPSMA T-BsAbs. Similar results were observed in in vivo Xenograft models of prostate cancer. Strikingly, CD3xPSMA bispecifics containing a novel low affinity anti-CD3 domain produced similar levels of tumor cytotoxicity compared to those with a traditional high affinity anti-CD3 domain, but with reduced cytokine production. Conclusions: We have created novel CD3xPSMA bispecific antibodies incorporating both monovalent and biparatopic anti-PSMA binding domains that mediate T-cell killing of PSMA+ tumor cells with minimal production of cytokines. Such T-BsAbs may improve safety, efficacy, and opportunities for combination therapy to treat CRPC.

Download Full-text

A novel T-cell bispecific antibody platform for efficient T-cell mediated killing of tumor cells with minimal cytokine release.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.209 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 209-209 ◽

Cited By ~ 1

Author(s):

Udaya Rangaswamy ◽

Andrew Boudreau ◽

Ben Buelow ◽

Starlynn Clarke ◽

Kevin Dang ◽

...

Keyword(s):

T Cell ◽

Tumor Cells ◽

Tumor Antigen ◽

Bispecific Antibodies ◽

Antigen Binding ◽

Human Pbmcs ◽

Cytokine Release ◽

In Vivo Efficacy

209 Background: Bispecific antibodies that recruit cytotoxic T cells to kill tumor cells are popular due to their targeted mechanism of action. Despite their attractiveness, there are limitations in the clinic due to undesirable toxicities associated with cytokine release. We describe here a platform for generation of a large collection of human anti-CD3 antibodies obtained from custom transgenic rats. Combining these unique anti-CD3 arms with different tumor targeting arms enables creation of bispecific antibodies of varying tumor cell killing capability. These anti-CD3 arms were combined with tumor antigen binding arms, namely, an anti-BCMA arm for multiple myeloma therapy, or an anti-CD22 arm for B-cell acute lymphoblastic leukemia therapy. Methods: Our platform utilizes a discovery approach involving antibody repertoire deep sequencing, high-throughput gene assembly, and recombinant expression, generating a highly diverse panel of antibodies with varying affinities. The CD3 antibodies were tested in in vitro T cell assays using human PBMCs to measure activation and cytokine release. Bispecific antibodies were evaluated for their ability to kill target cell lines upon co-culture with primary human PBMCs. The in vivo efficacy of bispecific antibodies was evaluated in a xenograft mouse model. Results: The in vitro T cell activity of these antibodies as measured by interleukin-2, interferon gamma levels and upregulation of the activation marker CD69 covered a broad spectrum of EC50 values. In co-culture systems with human PBMCs, anti-BCMA or anti-CD22 bispecific antibodies potently killed their respective target expressing cells with varying strengths. Additionally, the cytokine release from T cell activation correlated with the affinity of the anti-CD3 arms. The in vivo efficacy of the bispecific antibodies in a xenograft model with human PBMCs transferred into NSG mice showed striking tumor clearance at a wide range of doses. Conclusions: Our platform is highly suitable for creation of an extensive collection of bispecific antibodies for a variety of disease models by selecting the ideal anti-CD3 arm for each unique tumor antigen binding arm.

Download Full-text

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning

Scientific Reports ◽

10.1038/s41598-021-93842-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lauric Haber ◽

Kara Olson ◽

Marcus P. Kelly ◽

Alison Crawford ◽

David J. DiLillo ◽

...

Keyword(s):

T Cell ◽

Tumor Cells ◽

Binding Affinity ◽

T Cell Activation ◽

Cell Activation ◽

Therapeutic Index ◽

Bispecific Antibodies ◽

Cytokine Release

AbstractT-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

Download Full-text

CD18 Antibody Application Blocks Unwanted Off-Target T Cell Activation Caused by Bispecific Antibodies

Cancers ◽

10.3390/cancers13184596 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4596

Author(s):

Joseph Kauer ◽

Fabian Vogt ◽

Ilona Hagelstein ◽

Sebastian Hörner ◽

Melanie Märklin ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Lymphoid Cells ◽

Bispecific Antibodies ◽

Target Cells ◽

Cytokine Release ◽

Life Threatening ◽

Blocking Antibodies

T cell-recruiting bispecific antibodies (bsAbs) are successfully used for the treatment of cancer. However, effective treatment with bsAbs is so far hampered by severe side effects, i.e., potentially life-threatening cytokine release syndrome. Off-target T cell activation due to binding of bispecific CD3 antibodies to T cells in the absence of target cells may contribute to excessive cytokine release. We report here, in an in vitro setting, that off-target T cell activation is induced by bsAbs with high CD3 binding affinity and increased by endothelial- or lymphoid cells that act as stimulating bystander cells. Blocking antibodies directed against the adhesion molecules CD18/CD54 or CD2/CD58 markedly reduced this type of off-target T cell activation. CD18 blockade—in contrast to CD2—did not affect the therapeutic activity of various bsAbs. Since CD18 antibodies have been shown to be safely applicable in patients, blockade of this integrin holds promise as a potential target for the prevention of unwanted off-target T cell activation and allows the application of truly effective bsAb doses.

Download Full-text

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo

Cancer Immunology Immunotherapy ◽

10.1007/bf01517350 ◽

1995 ◽

Vol 40 (3) ◽

pp. 182-190 ◽

Cited By ~ 9

Author(s):

M. Belen Moreno ◽

Julie A. Titus ◽

Michael S. Cole ◽

J. Yun Tso ◽

Nhat Le ◽

...

Keyword(s):

Breast Cancer ◽

T Cell ◽

Bispecific Antibodies ◽

Cell Cytotoxicity ◽

T Cell Cytotoxicity

Download Full-text

Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽

10.1182/blood-2019-128288 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1934-1934 ◽

Cited By ~ 3

Author(s):

Eduardo Huarte ◽

Roddy S O'Connor ◽

Melissa Parker ◽

Taisheng Huang ◽

Michael C. Milone ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cytokine Release ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

Download Full-text

CD3-Activating Bi-Specific Antibody Targeting CD19 on B Cells in Mono- and Bi-Valent Format

Blood ◽

10.1182/blood-2018-99-112819 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4169-4169 ◽

Cited By ~ 2

Author(s):

Yumin CUI ◽

Zhihua Huang ◽

Xinfeng Zhang ◽

Wuzhong Shen ◽

Hanyang Chen ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

T Cell Activation ◽

Specific Antibody ◽

Cell Activation ◽

Cell Killing ◽

Cell Binding ◽

Dose Dependent

Abstract Immunotherapies targeting B-lineage-specific surface marker CD19 had demonstrated promising clinical results. Two CD19 CAR-T therapies (Kymriah® and Yescarta®) have been approved by FDA to treat patients with B cell malignancies, however, the complicated manufacturing process and low throughput limit its accessibility to more patients, especially in developing countries. The first CD3-activating bi-specific antibody targeting CD19, Blincyto, or CD19 BiTE, was approved to treat relapsed and refractory acute lymphoblastic lymphoma (r/r ALL). The relatively short half-life of Blincyto requires continuous IV infusion for weeks to maintain a steady levels of drug exposure, not to mention the high risk of developing severe cytokine release syndrome in patients. We had established a bispecific antibody platform ITabTM (immunotherapy antibody) for the generation of CD3-activating bi-specific antibodies that could potentially overcome the shortcomings of BiTEs. A CH1 domain was introduced into the ITabTM construct design with the intent to increase the molecular weight thus led to extend the serum half-life of the bispecific antibody. A novel CD3-activating and monkey cross-reactive antibody was generated with a less degree of T cell activation and cytokine release compared to BiTEs. A bi-valent binding to tumor associated antigen (TAA) format was established to target tumor cells and/or stem cells expressing very low levels of TAA. We report here the biological properties of the mono-valent/bi-valent binding of CD19 bi-specific antibody with CD3-activating activity (A-319/A-329). A series of studies were conducted to evaluate the bioactivities of A-319/A-329 in vitro and in vivo including binding to CD3 and CD19 antigens, T-cell and B-cell binding activities, T cell activation and proliferation and B cell killing activities in vitro as well as in vivo efficacy using human PBMC engrafted mouse xenograft models. The in vitro data showed that the mono-valent and bi-valent CD19 binding had little effect on the CD3-associated activities including CD3 antigen binding affinity, T cell binding and T cell activation. In contrast, the bi-valent binding format A-329 showed better potency compared to the mono-valent format A-319 in CD19 binding (KD 0.89 nM vs 19.4 nM); B cell binding (EC50 at 2.3 pM vs 462 pM); in vitro human B cell killing (EC50 0.2 pM vs 3.4 pM). Both A-319 and A-329 were capable of mediating tumor cell lysis with EC50 at 0.03~4 pM. A-329 demonstrated a greater killing activity on Pfeiffer, a human diffuse large B-cell lymphoma (DLBCL) cell line with a low expression of CD19 antigen. In human PBMC engrafted NOG mouse xenograft model, a dose-dependent tumor growth inhibition was observed at 0.5~100 µg/kg in both A-319 and A-329. In monkey studies, when A-319 and A-329 was dosed at 3, 10, 30 µg/kg, twice or three times weekly via IV infusion for A-329 or A-319. Dose-dependent elimination of peripheral blood B cells were observed with both ITabTM. The CD19 bi-valent format of A-329 revealed more complete B cell killing in monkeys. No significant difference of cytokine induction or liver injuries were observed between A-319 and A-329. These results demonstrated that both A-319 and A-329 may benefit patients with B cell malignancies with less dosing frequency and lower cytokine inductions especially, A-329 may have the potential to targeting the low CD19 expressing tumor stem cells. Disclosures No relevant conflicts of interest to declare.

Download Full-text

An optimal anti-tumor response by a novel CEA/CD3 bispecific antibody for colorectal cancers

Antibody Therapeutics ◽

10.1093/abt/tbab009 ◽

2021 ◽

Author(s):

Ninghai Wang ◽

Harshal Patel ◽

Irene Schneider ◽

Xin Kai ◽

Avanish K Varshney ◽

...

Keyword(s):

T Cell ◽

Binding Affinity ◽

Tumor Antigen ◽

Xenograft Model ◽

Variable Region ◽

Dependent Manner ◽

Cytokine Release ◽

Complex Development

Abstract Background CD3-based bispecific T cell engagers (bsTCEs) are one of the most promising bispecific antibodies for effective cancer treatments. To elicit target-specific T cell-mediated cytotoxicity, these bsTCEs contain at least one binding unit directed against a tumor antigen and another binding unit targeting CD3 in T cell antigen receptor complex. Development of CD3-based bsTCEs, however, has been severely hampered by dose limiting toxicities due to cytokine release syndrome. To address this limitation, we developed a novel functionally trivalent TCE (t-TCE) antibody containing affinity reduced CD3 binding unit, positioned to ensure monovalent CD3 engagement, in combination with bivalent tumor antigen binding of Carcinoembryonic Antigen (CEA). Methods We modeled the variable region of anti-CD3 in the CDRs of the heavy chain and obtained CD3 binders with reduced binding affinity. Two optimized versions CEA/CD3-v1 and CEA/CD3-v2 were identified and generated in tetravalent format, characterized and compared in vitro and in vivo. Results Our lead candidate, CEA/CD3-v2, demonstrated sub-nanomolar binding and picomolar potency against a panel of CEA-expressing cancer cell lines. In addition, we detected reduced T cell cytokine release with potent cytotoxic activity. Our t-TCE CEA/CD3-v2 molecule demonstrated strong anti-tumor effect in a dose dependent manner in human PBMC xenograft model. Furthermore, combination of CEA/CD3-v2 with atezolizumab provided synergistic antitumor effect. Conclusions Because of effective tumor cell killing with various level of CEA expression and reduced cytokine release, CEA/CD3 BsTCE may greatly benefit in CEA positive cancer immunotherapy. Statement of Significance. Through optimization of CD3 binding affinity and tetravalent format with functional monovalent binding to CD3, t-TCE CEA/CD3–2 molecule not only retains high potency in vitro and in vivo, but also significantly reduces cytokine release.

Download Full-text

Combinatorial targeting of multiple myeloma by complementing T cell engaging antibody fragments

Communications Biology ◽

10.1038/s42003-020-01558-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Maria Geis ◽

Boris Nowotny ◽

Marc-Dominic Bohn ◽

Dina Kouhestani ◽

Hermann Einsele ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Specific Immunotherapy ◽

Antibody Fragments ◽

Cytokine Release ◽

Major Drawback ◽

Single Antigen

AbstractBispecific T cell engaging antibodies (BiTEs) address tumor associated antigens that are over-expressed on cancer but that can also be found on healthy tissues, causing substantial on-target/off-tumor toxicities. To overcome this hurdle, we recently introduced hemibodies, a pair of complementary antibody fragments that redirect T cells against cancer-defining antigen combinations. Here we show that hemibodies addressing CD38 and SLAMF7 recruit T cells for the exquisite elimination of dual antigen positive multiple myeloma cells while leaving single antigen positive bystanders unharmed. Moreover, CD38 and SLAMF7 targeting BiTEs, but not hemibodies induce massive cytokine release and T cell fratricide reactions, a major drawback of T cell recruiting strategies. Together, we provide evidence in vitro and in vivo that hemibodies can be developed for the effective and highly specific immunotherapy of multiple myeloma.

Download Full-text